Ribonucleotide reductases (RNRs) convert nucleotides to deoxynucleotides in all organisms, thereby holding a central role in the DNA replication and repair. Very little is known of the assembly of Class la RNR's metallo-cofactor, a diferric-tyrosyl radical, which resides in the protein's [unreadable]2 subunit. The goal of the proposed research is to understand in particular how the iron of this cofactor is correctly loaded into [unreadable]2. Knowledge of the assembly of these diiron non-heme, non-iron-sulfur clusters, is not very well-understood at large, though this motif is found in proteins of considerable interest (i.e. methane monooxygenase). There are two parts to the proposal: a focused study towards identifying YfaE as a possible iron chaperone, and a broader effort to demonstrate the cell's utilization of iron homeostatic machinery in order to synthesize active cofactor. For the first goal, the specific aims include: A1) determining that transfer of iron between postulated chaperone YfaE and apo-[unreadable]2 is direct, and does not include a dissociation/reassociation mechanism;A2) obtaining heretofore unisolated [4Fe4S]-YfaE, both in dimer and monomer forms and determining if iron delivery to apo-[unreadable]2 efficacy is improved;A3) confirming binding between [unreadable]2 and YfaE through isothermal titration calorimetry;A4) testing the involvement of YfaE's cysteines 72 and 73 in the delivery of iron by generating serine and alanine mutants. For the second goal, the specific aims include: B1) generating iron transporter knockouts in E. coli;62) overexpressing RNR's [unreadable]2 subunit in these knockouts under varying growth conditions and monitoring cofactor generation by whole cell EPR spectroscopy;and B3) growing cells with "Fe-enriched ferritin FtnA, inducing overexpression of [unreadable]2, and monitoring iron migration by whole cell Mossbauer spectroscopy. PUBLIC HEALTH RELEVANCE: The cofactor of the ubiquitous Class la ribonucleotide reductase has already been effectively targeted in the successful killing of cancer cells and HIV. The current strategy of therapeutic agents has been to shut down the active cofactor, but an additional strategy could be to simply prevent cofactor assembly to begin with. Not much is known of how the cofactor is made;the research proposed focuses on identifying the iron- delivery agents needed for assembly. Knowledge obtained from these investigations will yield new strategies for the development of effective antibiotics, and novel anti-cancer treatments.